This invention relates to a novel asymmetrically functional membrane possessing an asymmetrical stratal structure and combining a function of selective gas permeation and a function of enzymatic reaction and to a method for the manufacture thereof. More particularly, this invention relates to an asymmetrical porous fluorine type resin membrane of a structure consisting of a hydrophilic layer and a hydrophobic layer, to an asymmetrically functional membrane obtained by immobilizing an enzyme in the hydrophilic layer of said asymmetrical membrane thereby imparting said membrane with both a function of enzymatic reaction and a function of selective permeation of gas, and to a method for the manufacture thereof.
The background of this invention will now be outlined in the following.
Enzyme-immobilized membranes are useful as sensing elements for enzyme electrodes, for example. For these membranes to give reliable response and produce consistent results, they must be manufactured in constant activity, thickness and configuration. Moreover, the membranes must be capable of high-speed response in order to permit rapid analysis and must have sufficient mechanical strength to provide stable continuous operation over prolonged periods.
An enzyme electrode consists of an enzyme-immobilized membrane and an electrochemical device. Typical electrochemical devices commonly used in the enzyme electrode are such selective electrodes as the ammonia gas selective electrode, the carbon dioxide gas selective electrode and the oxygen selective electrode. Basically, an enzyme electrode using these electrochemical devices needs at least two separate membranes of contrasting properties, i.e., a hydrophobic gas permeable membrane (usually a porous polytetrafluoroethylene membrame) on one hand, and an enzyme immobilized hydrophilic membrane on the other hand. The need for the employment of two membranes leads to various drawbacks with respect to response time, simplicity of electrode construction and long-term stability. It is clear that sufficiently close contact of the two membranes on a sensing end of an electrochemical device is prerequisite for a rapid electrode response. However, with two membranes, it is rather tedious and time consuming to construct the electrode in such a manner as the two membranes and the electrochemical devices come into close contact with each other, e.g., small air bubbles are often trapped between the two membranes and/or the membrane and the electrochemical device, leading to a sluggish electrode response. Moreover, the contact tends to loosen with time, due to differences in their mechanical properties. This results in a gradual slow down of the response time, and replacement of the enzyme membrane with another one is needed even if the enzyme activity remains unaffected.
These drawbacks may be avoided by direct formation of an enzyme membrane over a gas permeable membrane. For example, Mascini (M. Mascini. et al., Anal. Chem., 49, 795 (1977)) and Anfalt (T. Anfalt, et al., Anal Lett., 1973, 969) separately tried direct formation of an enzyme membrane on a porous membrane of polytetrafluoroethylene. The composite membrane obtained in this case is not practical because the enzyme membrane and the polytetrafluoroethylene resin adhere to each other with extremely weak strength and they readily separate from each other during use. Schultze (Schultze et al., Trans. Am. Soc. Artif, Intern. Organs XXV, p 66 (1979)) treated the surface of polytetrafluoroethylene membrane with an etching solution consisting of metallic sodium and an organic solvent then directly formed a gel membrane of glucose oxidase-albumin on the treated surface. Although this method enables the polytetrafluoroethylene membrane and the enzyme membrane to adhere to each other with greater strength than the method described above, the composite membrane obtained by this method is unstable and unsuitable for practical application since separation of the membranes cannot be prevented because the enzyme membrane of low mechanical strength is simply superposed on the surface of the polytetrafluoroethylene membrane without any protection. Further by this method, it is difficult to reproducibly obtain an enzyme membrane of fixed thickness at all times. Moreover, since the treatment with the etching solution alters the polytetrafluoroethylene membrane configuration, it is difficult to obtain treated membranes having constant response characteristics. Therefore, the produced composite membrane is not suitable for use in a sensor which is particularly required to maintain constant response characteristics even when the composite membrane is replaced with a new one.
As a membrane superior to those obtained by the above described methods, there has been developed an asymmetrically functional membrane which is produced by immobilizing an enzyme within continuous micropores distributed to a fixed depth d in the direction of thickness in a porous fluorine type resin membrane of a thickness of D (d&lt;D) thereby enabling the layer of thickness d of the membrane to serve as an enzyme-immobilized membrane having a function of enzymatic reaction and the remaining layer of thickness D-d of the membrane to serve as a membrane having a function of selective gas permeation. Even if, in this case, the enzyme membrane is in the form of mechanically weak gel, it acquires amply high mechanical strength because it is embedded in the strong matrix of the fluorine type resin membrane and thus is protected by the matrix. This asymmetrical functional membrane is superior to the earlier developed membranes produced by joining separately produced enzyme-immobilized and fluorine type gas permeable membranes since it allows a membrane of the same function to be obtained by processing only a single membrane. In the production of such a membrane, however, it is necessary to cause an enzyme solution to permeate the continuous micropores of the fluorine type resin membrane in order to immobilize the enzyme in the membrane. However, since, the membrane being hydrophobic, it is extremely difficult to incorporate the enzyme solution into the micropores. For successful immobilization of the enzyme, therefore, the fluorine type resin membrane must be given a treatment for making the resin hydrophilic without changing micropore structure of the membrane.
Various methods have been available for making a fluorine type resin hydrophilic. These include, for example, treatment with a metallic sodium-organic solvent (or ammonia) system, graft reaction by the use of radiation, a method involving use of an organic titanium compound, adsorption treatment by the use of a surface active agent or a hydrophilic high molecular compound, plasma treatment, and corona-discharge treatment. When any of these known methods is applied to a porous fluorine type resin membrane having a thickness on the order of 20 to 100 .mu.m, it is only possible to make the membrane hydrophilic throughout the entire thickness thereof or to make it hydrophilic only to a small depth from the treated surface. None of these conventional methods has been able to provide a sophisticated treatment enabling the membrane to be made hydrophilic only to a desired thickness without changing the original micropore structure. When the membrane is made hydrophilic throughout the entire thickness, it is deprived of its function of selective gas permeation and is no longer useful as an enzyme electrode. When the membrane is rendered hydrophilic only in its surface region, it is incapable of stably immobilizing an ample amount of enzyme for the membrane to produce sufficiently large output.
One object of this invention is to provide an asymmetrically functional membrane of fluorine type resin having a desired thickness thereof selectively made hydrophilic.
Another object of this invention is to provide an asymmetrically functional membrane of fluorine type resin which combines a function of enzymatic reaction and a function of selective gas permeation.
Yet another object of this invention is to provide a method for the manufacture of an asymmetrically functional membrane of fluorine type resin which combines a function of enzymatic reaction and a function of selective gas permeation.